Tr a n s l a t i o n a l O n c o l o g y
Volume 3 Number 2
pp. 109–113 109
Circulating miR-210 as a Novel Hypoxia Marker in Pancreatic Cancer1
Allen S. Ho*,2, Xin Huang†,2, Hongbin Cao†, ‡ Claudia Christman-Skieller†, Kevin Bennewith , Quynh-Thu Le†, and Albert C. Koong† *Department of Otolaryngology–Head & Neck Surgery, Stanford University School of Medicine, Stanford, CA, USA; †Department of Radiation Oncology, Center for Clinical Science Research, Stanford University School of Medicine, Stanford, CA, USA; ‡Integrative Oncology Department, BC Cancer Research Centre, Vancouver, British Columbia, Canada V5Z 1L3
Abstract MicroRNA are small noncoding transcripts involved in many cellular mechanisms, including tumorigenesis. miR-210, in particular, is induced by hypoxia and correlates with adverse outcomes in certain cancers. Because pancreatic adenocarcinomas exhibit extremely hypoxic signatures, we hypothesized that miR-210 may serve as a diagnostic marker for screening or surveillance for pancreatic cancer. Plasma samples were obtained from newly diagnosed pancreatic cancer patients and age-matched noncancer controls. miRNA was extracted directly from plasma and reverse-transcribed to complementary DNA. A known quantity of synthetic Caenorhabditis elegans miR-54 (celmiR-54) was added for normalization. miR-210 and cel-miR-54 were then measured using quantitative reverse transcription polymerase chain reaction. An initial cohort of 11 pancreatic cancer patients and 14 age-matched controls was used as the test set and a second cohort of 11 pancreatic cancer patients and 11 controls was used as the validating set in this study. miR-210 was reliably detected and quantified, with a statistically significant four-fold increase in expression in pancreatic cancer patients compared with normal controls (P < .00004) in the test set. This difference was confirmed in the validation group (P < .018). In summary, circulating miR-210 levels are elevated in pancreatic cancer patients and may potentially serve as a useful biomarker for pancreatic cancer diagnosis. Translational Oncology (2010) 3, 109–113
Introduction Pancreatic cancer remains one of the most lethal malignancies, with most cases diagnosed after metastatic spread. Median survival in patients with locally advanced/metastatic disease is less than 12 months, with overall 5-year survival less than 5% despite aggressive multimodality treatment . Tumor markers may facilitate earlier diagnosis and have the potential for use in monitoring response to cancer therapies, but there is no current biomarker that reliably serves this purpose. There is, therefore, a tremendous need to identify novel noninvasive biomarkers for early tumor detection. MicroRNA (miRNA), a class of naturally occurring, noncoding small RNA, regulate the expression of most genes. On a molecular level, miRNA destabilize messenger RNA by repressing translation and shortening the polyA tail. On a cellular level, miRNA play critical roles in differentiation, proliferation, apoptosis, and metabolism and have ultimately been linked to cancer development . Their as-
sociation with tumorigenesis has given rise to their potential for clinical diagnosis and as therapeutic targets. Hypoxia, or low oxygen, is an essential feature of the tumor microenvironment, particularly in pancreatic cancer . Cancers with increased hypoxia exhibit poorer prognosis and greater resistance to chemotherapy and radiation. Hypoxia has also been demonstrated to induce differential miRNA expression [4–7]. Several investigators have Address all correspondence to: Albert C. Koong, MD, PhD, Department of Radiation Oncology, Stanford University School of Medicine, 269 Campus Dr West, CCSR1245C, Stanford, CA 94305-5152. E-mail: [email protected]
1 This study was supported by PO1 CA67166 (A.C.K., Q.T.L., and X.H.) and R01 CA118582 (Q.T.L. and H.C.). The authors have no potential conflict of interest. 2 These coauthors contributed equally to the article. Received 27 August 2009; Revised 2 December 2009; Accepted 9 December 2009 Copyright © 2010 Neoplasia Press, Inc. All rights reserved 1944-7124/10/$25.00 DOI 10.1593/tlo.09256
miR-210 and Hypoxia in Pancreatic Cancer
Ho et al.
Translational Oncology Vol. 3, No. 2, 2010
reported that miR-210, in particular, is increased in response to hypoxia [5,6,8,9] and may in fact be the principal miRNA expressed in a number of different cancer types through a hypoxia-responsive element . Interestingly, miR-210 induction is regulated by hypoxia-inducible factor 1α (HIF-1α), and it serves as an important regulator for inhibiting DNA repair pathways and promoting genomic instability . Furthermore, miR-210 facilitates the mRNA degradation of normoxic genes, providing another mechanism of HIF-regulated gene expression . In the present study, we assessed the potential of circulating miR-210 to function as a diagnostic marker for pancreatic cancer. On the basis of previous work by Mitchell et al.  and Chen et al. , we have developed a plasma-based assay that reliably quantifies miR-210 from archived patient plasma samples. Here we report that plasma miR-210 expression from patients with newly diagnosed locally advanced pancreatic adenocarcinomas was significantly elevated in comparison to age-matched controls, using a test and a validating set. Patients and Methods
Cohorts Plasma samples for pancreatic cancer patients and controls in cohort 1 were collected and archived between June 2006 and January 2008, whereas plasma samples for cohort 2 were collected and archived between March 2005 and October 2006. All samples were collected before any treatment, and miR-210 levels were analyzed retrospectively from these groups. All patients were identically staged with pancreatic protocol computed tomographic scans and evaluated at the Stanford gastrointestinal multidisciplinary tumor board. All patients had locally advanced, unresectable stage T4 disease. Recurrent malignancies were excluded from the study. The protocol described was first tested on a cohort of 11 pancreatic cancer patients and 14 age-matched healthy controls (cohort 1; Table 1). A second validation set of 11 pancreatic cancer patients and 11 age-matched healthy controls (cohort 2; Table 2) was used to confirm our initial results and to achieve greater statistical power. There was no statistically significant difference in
plasma miR-210 levels between the two control groups (2-tailed t-test, P = .22).
Plasma Extraction Five to ten milliliters of whole blood was obtained from each patient. Plasma was extracted by centrifuging whole blood at 3000 rpm for 10 minutes at room temperature and then frozen as separate aliquots at −80°C for storage. One hundred microliters of thawed plasma was boiled for 10 minutes at 100°C and then centrifuged at 13,000g for 10 minutes at 4°C. Approximately 1.1 μl of supernatant was collected.
Complementary DNA Production To normalize by volume, synthetic Caenorhabditis elegans miR-54 (cel-miR-54) (Integrated DNA Technologies, Coralville, IA) served as a control. cel-miR-54 has previously been shown not to affect human miRNA detection . Complementary DNA was generated by mixing the extracted RNA (1.1 μl) with 5× miR-210 primer (1.0 μl), 5× cel miR-54 primer (0.9 μl), 0.25 nM cel miR-54 oligo (0.1 μl), 5× First Strand buffer (1.0 μl), dithiothreitol (0.5 μl), 25 mM dNTP (0.2 μl), RNAse Out Inhibitor (0.1 μl), and reverse transcriptase (0.1 μl). This mixture was assayed on a standard polymerase chain reaction (PCR) thermal cycler at 16°C for 30 minutes, at 42°C for 30 minutes, at 85°C for 5 minutes, and then at 4°C afterward.
Quantitative Reverse Transcription–PCR Quantitative reverse transcription–PCR (qRT-PCR) was performed using an ABI 7900 Thermal Cycler, with miR-54 and miR-210 quantified using TaqMan MicroRNA Assays (Applied Biosystems, Foster City, CA). Twenty-five microliters of dH2O was mixed with 5 μl of complementary DNA. Four microliters of this dilution was mixed with 2× Master Mix (5 μl), 20× PCR Primer (0.5 μl), and dH2O (0.5 μl) before being pipetted onto a 394-well plate. qRT-PCR was then performed at 50°C for 2 minutes, at 95°C for 10 minutes, at 95°C for 15 seconds, and at 60°C for 60 seconds, with the last two steps repeated for a total of 40 cycles. Each reaction was performed in triplicate.
Table 1. Cohort 1. Cohort 1
CA 19-9 (U/ml)
miR-210 Expression (Mean ± SD)
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 P11 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14
85 60 60 68 65 57 60 63 51 79 46 64 59 58 61 49 58 66 62 55 58 51 61 62 60
Head Head Head Head Head Head Body Head Body Body Head
24 243 235 4514 1270